Apparatus to measure multiple signals from a liquid sample

ABSTRACT

One or more homogenizing elements are employed in a flow through, multi-detector optical measurement system. The homogenizing elements correct for problems common to multi-detector flow-through systems such as peak tailing and non-uniform sample profile within the measurement cell. The homogenizing elements include coiled inlet tubing, a flow distributor near the inlet of the cell, and a flow distributor at the outlet of the cell. This homogenization of the sample mimics plug flow within the measurement cell and enables each detector to view the same sample composition in each individual corresponding viewed sample volume. This system is particularly beneficial when performing multiangle light scattering (MALS) measurements of narrow chromatographic peaks such as those produced by ultra-high pressure liquid chromatography (UHPLC).

PRIORITY

This application is a continuation of U.S. patent application Ser. No.16/566,827, filed Sep. 10, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/761,808, filed Mar. 20, 2018, and claimspriority to U.S. Provisional application No. 62/221,879, filed Sep. 22,2015, and to PCT Application No. PCT/US2016/052955, filed Sep. 21, 2016.

RELATED PATENTS

The following references related to the background and application ofthe present invention are hereby incorporated by reference:

-   Steven P. Trainoff, U.S. Pat. No. 7,386,427 B2, issued 10 Jun. 2008,    “Method for correcting the effects of interdetector band    broadening”;-   Steven P. Trainoff, U.S. Pat. No. 7,911,594 B2, issued 22 Mar. 2011,    “Method to derive physical properties of a sample after correcting    the effects of interdetector band broadening”;-   Gary R. Janik, et al., U.S. Pat. No. 5,900,152, issued 4 May 1999,    “Apparatus to reduce inhomogeneities in optical flow cells”;-   Gary R. Janik, et al., U.S. Pat. No. 5,676,830, issued 14 Oct. 1997,    “Method and apparatus for reducing band broadening in    chromatographic detectors”;-   Steven D. Phillips, et al., U.S. Pat. No. 4,616,927, issued 14 Oct.    1986, “Sample cell for light scattering measurements”;-   Raymond P. W. Scott and Elena Katz, U.S. Pat. No. 5,032,183, issued    16 Jul. 1991, “Low dispersion fluid conduit useful in chromatography    systems”; and-   Steven P. Trainoff, U.S. Pat. No. 7,982,875, issued 19 Jul. 2011,    “Improved method and apparatus for measuring the scattered light    signals from a liquid sample.”

Definitions

By “particle,” we refer to such objects as protein and polymer moleculestogether with their conjugates and co-polymers, viruses, bacteria,virus-like particles, liposomes, polystyrene latex particles,nanoparticles, and all such particles within the approximate size rangeof one to a few thousand nanometers.

The terms quasi-elastic light scattering (QELS), dynamic lightscattering (DLS), and photon correlation spectroscopy (PCS) arefrequently used to describe the same phenomenon, namely the measurementof scattered light from particles undergoing Brownian motion. In thisspecification we will use the term QELS, but it should be noted thatQELS is equivalent to the other mentioned terms often used in the art.QELS stands distinct from other light scattering measurement techniques,the most common of which is multi-angle light scattering (MALS),formerly referred to as “Differential Light Scattering,” which measuresthe angular dependence of light scattered from a solution of particles.

Throughout this specification reference will be made to opticalmeasurement cells. There are, in the most general form, two cell typesthat will be discussed herein. The first is a cell, such as thatdisclosed in U.S. Pat. No. 4,616,927, wherein the direction of flow ofthe liquid sample and solvent through the cell runs along the same pathessentially parallel to the illuminating beam. This cell configurationwill be referred to as a “parallel cell.” An alternative cell designdiscussed herein is one wherein the fluid flow runs transverse to theilluminating beam. This cell configuration will be referred to as a“perpendicular cell.” This is the traditional structure used formeasurement of light scattered from a cylindrical container. It shouldbe noted that this nomenclature should not be indicative of a strictlimitation, but rather provide a general description and designationonly. For example, the beam traversing a parallel cell may be slanted ata small angle, such that at the entrance of the cell the beam almostgrazes the bottom of the bore, while at the exit it almost grazes thetop of the bore, and still be considered a “parallel cell” even thoughthe flow and the beam are not precisely parallel. Indeed, in severalembodiments of the invention, it may be desirable to direct the beamthrough the sample at an angle in order to minimize reflections, reducenoise, etc. The optical measurement cells discussed above and throughoutthe specification are flow through cells which contain a bore throughwhich the liquid sample passes. While these bores are generally of acircular or near circular cross section, the sample bore should not beconsidered limited to this shape. The present invention is applicable tocells with bores cross sections in a variety of shapes, includingrectangular, half-circular, elliptical, triangular, etc., as well asirregular shape in cross section and path.

There are many means by which particles of various sizes, masses andcharges in a liquid sample may be separated from their constituents. Inthis disclosure we will focus primarily on a technique generallyreferred to as ultra-high pressure liquid chromatography (UHPLC), whichis also known as ultra-high performance liquid chromatography, howeverit should be noted that most separation techniques used today arespecies under the genus liquid chromatography, and all liquidchromatography techniques can benefit from the invention disclosedherein. Therefore, while the discussion below will primarily refer toUHLPC (sometimes also referred to by the trade name UPLC®), as it is themethod which benefits most from the present invention, the invention isalso applicable to other liquid chromatography techniques such as highpressure liquid chromatography (HPLC), which is frequently referred toas size exclusion chromatography (SEC) or gel permeation chromatography(GPC), and reversed phase chromatography. In addition the invention isalso applicable to use with other flow based separation techniques, suchas field flow fractionation (FFF), wherein the separated sample isdelivered to the detection cell via a tube.

BACKGROUND

The analysis of macromolecular or particle species in solution isusually achieved by preparing a sample in an appropriate solvent andthen injecting an aliquot thereof into a separation system such as aliquid chromatography (LC) column or Field Flow Fractionation (FFF)channel wherein the different species of particles contained within thesample are separated into their various constituencies. Once separatedby such means, generally based on size, mass, or column affinity, thesamples are subjected to analysis by means of light scattering,refractive index, UV absorption, electrophoretic mobility, viscometricresponse, etc. In this disclosure we will primarily concern ourselveswith multi-angle light scattering (MALS).

A typical HPLC-MALS setup is shown in FIG. 2. Solvent is generally drawnby an HPLC pump 201 from a solvent reservoir 202 through a degasser 203and then pumped through filtering means 204 to the injection valve 211.A liquid sample 205 is injected into the sample loop 212 of theinjection valve 211, generally by a syringe 206. The sample, however,may be added to the flow stream by means of an auto injector rather thanthe manual means described. The fluid sample then flows from theinjector through one or more HPLC columns 207, where the molecules orparticles contained within the sample are separated by size, with thelargest particles eluting first. The separated sample then passessequentially through a MALS detector 208 and a concentration detectorsuch as a differential refractometer 209 before passing to waste. Otherinstruments capable of measuring the physical properties of themolecules or particles in the sample may also be present along the flowstream. For example, a UV/Vis absorbance detector and/or a viscometermight be present within the chain of instruments. In general, datagenerated by the instruments is transmitted to a computer that iscapable of collecting, storing, and analyzing the data and reporting theresults to the user.

As discussed above, a sample containing aliquot is injected into aseparation system, such as the HPLC system shown in FIG. 2, or, asrelates more closely to the present invention, a UHPLC system which issimilar but wherein the columns 207 are one or more UHPLC columns ratherthan HPLC columns, and the pump 201 is capable of producing the higherpressures at which UHPLC systems operate. The columns separate thesample by size into its constituent fractions. Each of these eluting andseparated fractions results in a “peak” which passes via tubing to ameasurement volume. Each detection instrument measures, in turn, asignal from the peak as it passes through the measurement volume. Asingle aliquot may generate any number of peaks, for example, amonodisperse sample of single particle size will generate only one peak.As each sample peak passes through a measurement volume a signal will bedetected that relates to the sample being analyzed at any given instantas it passes through the cell. These finite measurements are oftenreferred to as “slices,” each slice representing the instantaneousmeasurement of the sample being detected at a given volume of eluentflowing through the cell. These signals are digitized and stored in acomputer, and the resultant data is generally reported to the user as apeak 301, such as that shown in FIG. 3, wherein each element of the peakrepresents a given slice. The data at a given slice, in this case thelight scattering data over a plurality of angles, 302, corresponding toa single slice 302 a may be viewed and analyzed utilizing a softwareprogram such as ASTRA®. The data shown in FIG. 3 also includes thesignal 303 recorded from a differential refractometer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a geometry schematic of a MALS measurement.

FIG. 2 shows a typical HPLC-MALS configuration of the various elementsassociated with a MALS measurement.

FIG. 3 is an example of MALS and concentration data collected over anentire peak including MALS data displayed for a single slice.

FIG. 4 shows the parallel cell structure for light scatteringmeasurements and associated manifold system of U.S. Pat. No. 4,616,927.

FIG. 5 illustrates an alternative light scattering cell configurationthat contrasts in its flow path with that of the cell of FIG. 4.

FIG. 6 illustrates (a) a flowing sample subject to spreading due toPoiseuille flow, (b) the field of view of three misaligned detectorscollecting scattered light from a sample cell, and (c) the intensity vs.time plots illustrating the time shift seen by each detector due tomisalignment.

FIG. 7 shows examples flow profiles within a measurement cell due toDean flow from bent or improperly directed inlet tubing.

FIG. 8 shows the difference in time for a single concentration profilefront to pass two detectors that view slightly different detectionvolumes.

FIG. 9 shows a schematic representation of an embodiment of theinventive measurement system employing a helical, coiled inlet tube thatcorrects for asymmetrical flow profiles within the inlet tube.

FIG. 10 illustrates an embodiment of the inventive flow system with agraphical representation of the flow profile concentration contours of asample passing there through. Note this particular embodiment employs aflow distributor at both the inlet and the outlet of the measurementcell.

FIG. 11 shows a flow cell assembly employing an annular flow distributorin a parallel cell configuration.

FIG. 12 illustrates a preferred embodiment of a flow distributor for theinventive measurement system wherein a square shaped flow interrupter isseated within a circular gasket sandwiched between two meshed sheetsthat are in turn sandwiched between another pair of circular gaskets.

FIG. 13 shows a preferred embodiment of the invention utilizing manydisclosed elements including a pair of the flow distributor sandwichesof FIG. 12 held together in the flow cell manifold that also permits theconnection of coiled inlet and outlet tubes with standard fittings.

FIG. 14 presents a visualization of flow profiles in a measurement cellwherein the profiles show asymmetry due to Dean Flow caused by bentinlet tubing.

FIG. 15 illustrates the flow profile within the cell of a near idealconfiguration wherein the inlet tubing is perfectly straight prior tocoupling with the flow cell.

FIG. 16 illustrates asymmetry in the sample profile due to a 25 mm bendradius in the tubing prior to coupling with the flow cell.

FIG. 17 illustrates the sample profile with the same bend conditions asthose presented in FIG. 16, however, in this illustration, an embodimentof the invention wherein coiled inlet tubing and a mesh flow distributorare employed.

FIG. 18 contrasts two chromatograms wherein the detectors (a) are and(b) are not misaligned. Misalignment can result in significant errors.

A DETAILED DESCRIPTION OF THE INVENTION

MALS detection systems have frequently employed a flow cell wherein ahorizontal bore defines the region of both fluid flow and the path alongwhich the measurement beam traverses the cell. Such a cell was disclosedin U.S. Pat. No. 4,616,927. The cell, herein referred to as a parallelcell, is a section of a cylinder as shown in FIG. 4, with a bore drilledthrough a diameter. This cell design further acts as a lateral lens,such that the beam 401 acts as a line source near the detection region402 at which a plurality of detectors (not shown) positioned at variousangles around the circumference of the cell are directed to receivelight scattered therefrom by the sample. Therefore, while thisinnovative design allowed for the gathering of more light from thedetector at a given scattering angle, this benefit comes at the cost ofsome averaging of the sample concentration naturally occurring along thebreadth of the line source along the flow path at a given measured angledue to the larger sample volume required for this design. Another meansby which the amount of light scattered at a given angle is increased wasdisclosed in U.S. Pat. No. 7,982,875, is a modification to the celldisclosed in U.S. Pat. No. 4,616,927, where the sample cell acts as botha vertical and lateral lens.

The trend in the industry to lower sample volumes that producecorrespondingly lower volume peaks has driven the advent of Ultra HighPressure Liquid Chromatography (UHPLC). As a result MALS detectionsystems for measuring UHPLC peaks must appropriately scale insensitivity and volume. Liquid samples passing through the measurementcell, either the parallel cell as discussed above, or an alternativeconfiguration, such as that shown in FIG. 5(a), and referred to as aperpendicular cell, are greatly affected by the flow mechanics of thesample as it enters the cell. In the perpendicular cell, the sample flow501 enters the bore 502 of the cell structure 503 perpendicularly to thebeam 504. An array of detectors situated about the measurement cellcollect scattered light. Ideally each detector would view the sameilluminated volume, however, due to non-perfect alignment and variationin the geometry of the sample volume seen detector located at differentangles, they necessarily view slightly different volumes and thereforeit is essential that the sample be uniformly distributed throughout thecell. An example of the variability in the geometry of the scatteredvolume based on the field of view of detectors placed at differentangles is shown in FIG. 5(b). A laser beam 504 of diameter 2 r passesthrough the bore 502 of the measurement cell. Three detectors D1, D2 andD3 are placed about the cell and each is directed to detect scatteringfrom the illuminated sample near the center of the cell. However, eachof the three detectors has a unique field of view which results indetection regions whose corresponding observed scattering volumes d1,d2, and d3 vary. In this example detector D3, located at 90° to thelaser beam will have the smallest scattering volume, while detector D3,located at a low angle, will have the largest. If the sampleconcentration is uniform across all of the measured scattering volumesd1, d2, and d3, the signals may simply be normalized to unity. However,if the sample profile as it passes through the beam is non-uniform, notonly will each sample view a different sized scattering volume, but thevariation in the geometry of the scattering volumes will cause eachdetector to view different sample passing there through. It is thereforecritically important that the sample be homogeneous throughout the rangemeasured scattering volumes seen by individual detectors.

Issues of sample uniformity are particularly evident with UHPLC as thepeak widths are comparable to the sample volume of the measurement cell.Laminar flow along the capillary tubing results in Poiseuille flowduring transit of the separated sample from the UHPLC system to themeasurement cell. In Poiseuille flow the velocity profile is parabolicwhere the fluid flow at the wall of the tubing is zero, and the flow inthe center of the tube is at a maximum which is theoretically 1.5 timesthat of the average flow of a sample region. As shown in FIG. 6, thePoiseuille flow profile 601 causes a narrow sample peak 602 exiting theseparation system to be broadened over time as it passes through a bore,be it a length of tubing or a measurement cell. Under these conditionsany mild misalignment of the detectors wherein each detector views aslightly different illuminated volume can cause each detector to measurea slightly different section of the sample passing through the cell at agiven instant in time. For example, consider the sample profiles shownin FIG. 6a wherein the sample is traveling down a detection cell andbeam 603 traverses the cell at the point indicated as the sample, attime t=2, passes through. A cross section of the sample at the beaminterface is shown in FIG. 6b . At time t=2, the front of the sample hasreached a region indicated as section 604, whereas section 605 consistsof only solvent. Three different light scattering detectors are orientedtowards the center of the cell, but slight variations in alignment causethem to see different regions of the illuminated beam. Therefore thefirst detector 606 detects light scattered from a region 606 a, whereinpart of the beam within the detection area illuminates the sample 604.The second detector 607 sees a different region 607 a wherein light isscattered exclusively from the sample 604, and the third detector 608,at time t=2 sees no sample scattering at all. This effect causes a timeshift in the intensity peaks recorded for each a detector, the intensityvs. time plot for each detector is represented in FIG. 6c . It should benoted that if perfect alignment were achieved, that is, if each detectorviewed the exact same measurement volume, this time variation withrespect to intensity recorded at each detector would not be present inthe same manner, but measurements could still suffer from some temporalmisalignment of the peaks due to the different scattering volumesdefined by the intersection of the beam and the solid scattering angleviewed by each detector discussed above.

Dispersion, as the sample enters the measurement cell, exacerbated bythe phenomena of Dean Flow and convective diffusion, wherein radialnon-uniformity from slight bends in the inlet tubing cause the sample toenter the cell asymmetrically introducing error into the measurement dueto a time delay in the light scattering signal reaching the individualdetectors. It has been shown in literature, for example see A. Kaufman,et. al., “Extra-Column Band Spreading Concerns in Post-Column PhotolysisReactors for Microbore Liquid Chromatography,” Current Separations,17(1) 9-16 (1998), that peak dispersion in bent or coiled tubes can bedescribed by the dimensionless parameter Dn²Sc where Dn is the Deannumber

$\begin{matrix}{{Dn} = {{Re}\sqrt{\frac{r_{t}}{r_{c}}}}} & (3)\end{matrix}$

where r_(t) and r_(c) are the tube inner diameter and the tube bendradius respectively and Re the Reynolds number and Sc is the Schmidtnumber

$\begin{matrix}{{Sc} = \frac{\eta}{D_{m}\rho}} & (4)\end{matrix}$

where D_(m) is the sample diffusion coefficient, η the solvent viscosityand p the solvent density. It has been found that a Dn²Sc of less than100 has little impact on peak dispersion while greater values can have asignificant influence. FIG. 7 shows a sample with a Poiseuille profileentering in a measurement cell consisting of various bands of sampleconcentration. Each contour line 701, 702, 703, 704, 705 represents aboundary of a given sample concentration band. If the sample enters thecell through a tube that is perfectly parallel to the bore and with nocontribution from Dean Flow, r_(c)=∞, the sample boundaries will beconcentric at the plane defined by the bore of the sample cell where itis intersected by the illuminating beam 706 as shown in FIG. 7(b).However, any asymmetry caused, for example, by an improperly angled orconnected inlet tubing, or by radial non-uniformity as a result of DeanFlow from a curved tube, as indicated in FIG. 7(a) will cause a profilesuch as those shown in FIGS. 7(c) and 7(d). As discussed above, if weconsider a MALS detection system with three separate detectors orientedso as to detect light scattered from the illuminating beam by the sampleas it passes there through, we can see how, if each detector isperfectly aligned to the same region of the cell, each will “see” thesame band of the sample, to the extent to which the variation indetection geometry of each detector discussed above allows, even whenthe sample is asymmetrically distributed. However, perfect alignment,although desired is never practically achieved, and therefore, as waspreviously shown in FIG. 6, each detector will be measuring a differentregion of concentration as the sample passes through the illuminatingbeam. Therefore there will always be small variations in sample volumethat are within the field of view of each of the three detectors at agiven instant in time. This issue is further exacerbated with theaddition of more detectors.

The time shift between detectors is further explained by way of FIG. 8wherein a single concentration contour is shown and there are twodetection volumes defined by the field of view of two separatedetectors. In this case the detection volumes are labeled d₁, and d₂.The contour line 801 represents the boundary of a given concentration ofsample. The time at which the two detectors see the sample followingcontour line 801 varies depending on the lateral positioning of thedetector field of view. At time t=0, neither detector is seeing anilluminated volume of sample. The contour line 801 moves downward as thesample travels through the capillary between t=0 and t=2. At time t=1,D2 sees the sample following the contour 801, at time t=2, D1 sees thecontour, and therefore the time delay, Δt, is simply t₂-t₁. Again theintensity vs. time peak is shown graphically in FIG. 8 c.

Another issue complicating the accurate measurement of light scatteringfrom separated peaks involves peak tailing, which is a result of axialbroadening along the capillary and is caused directly by laminar flowwithin the tubing. The flow velocity at the edges of the tubing is zeroso any sample near this boundary can take a very long time to elute intothe measurement cell.

Therefore, in order to improve the reliability of MALS and other opticalmeasurements after separation, particularly UHPLC separation, all ofthese issues: peak tailing, non-uniform sample profile entry into themeasurement cell, and Poiseuille flow within the tubing and sample cell,may be addressed by the inventive methods and apparatus disclosedherein. This invention seeks to promote plug flow of the sample throughthe cell whenever possible and improve symmetry of the flow profilewithin the sample cell. Plug flow is a simple velocity profile of flowthrough a tube wherein each element across the tube diameter has thesame velocity. Therefore, plug flow stands in stark contrast toPoiseuille flow discussed above.

The first issue to be addressed is the non-uniformity of the samplewithin the tubing as it enters the flow cell. The present invention, oneembodiment of which is shown in FIG. 9, makes use of a length of coiledtubing 902 designed, as discussed below, to disrupt Poiseuille flow andcontributions due to Dean Flow prior to entry of the sample into theflow cell. The coiled tubing promotes radial mixing transverse to theflow path, creating, by the time the fluid enters the cell, an equaldistribution across the breadth of the inlet tubing, resulting in asymmetrical flow profile, with respect to the bore, to enter the flowcell. The coiled tubing also promotes Taylor dispersion causing shearflow to increase the effective diffusion coefficient of the sample awayfrom the tubing wall. This effect helps to mitigate peak tailing asdiscussed above. The narrow bore tube 902, generally made of stainlesssteel, is formed in a helix shaped coil wherein the turn radius of thetubing is preferably about 15 times the inner radius of the tubing.Although coiled tubing may be less efficient for this type of mixingthan one comprising a serpentine path, it is more easily manufacturedand maintained in a useful shape. After passage of the sample throughthe coiled tubing 902, it passes along the measurement cell through theilluminating beam to the cell outlet, where it passes to the outlet cellwhich may also be connected to a length of coiled tubing as the samplepasses to the next instrument in the chain or to waste. Surrounding thecell are a set of photo detectors which collect light scattered from thesample by the illuminating beam at a plurality of scattering angles.

In order to promote uniform sample flow within the sample cell itselfthe inventive apparatus may make use of a flow distributor placed in thepath of the incoming jet of fluid and/or a flow distributor placed afterthe outlet of the cell and prior to the outlet tubing. FIG. 10 shows across section of the elements of one embodiment of the presentinvention. The inlet tubing 1001 allows flow to enter an inlet entryvolume 1002, where it thereafter passes through a porous barrier actingas the inlet flow distributor 1003. There is an impedance mismatchbetween the inlet tube and the detection cell which is proportional tothe square of the average fluid velocity. ΔP₁ defines the pressuredifference required for the sample to fill the entire inlet entry volume1002. ΔP₂ defines the pressure difference required for the sample topass from the inlet entry through the flow distributor 1003 into thecell entry volume. In order for the flow to be properly distributedtransverse to the fluid flow through the cell, prior to entering thecell entry volume 1004, ΔP₂>>ΔP₁. The diameter of the detection cell,generally being on the order of 10 times the inner diameter of the inlettubing, would need to generate a backpressure 10,000 times greater thanthe nominal back pressure of the sample cell. When this condition isachieved, one may approximate the flow velocity through the distributor1003 as being uniform everywhere inside the volume 1002. An embodimentalso including a coil at the inlet results in the sample being uniformacross the orifice 1001. Given the uniform velocity through the flowdistributor 1003, the sample will distribute itself to be uniform acrossthe top surface achieving, to good approximation, plug flow.

For example, in one experimental configuration ΔP₁-ΔP₂ was measured at 2psi, this is 1000 times greater than the calculated requiredbackpressure which will only improve sample distribution as it entersthe measurement cell. After passing through the flow distributor 1003,the flow enters a cell entry volume 1004 and then passes into the innerbore 1005 of the measurement cell 1006. A light beam 1007 passes throughthe cell and photo detectors are placed in a ring around the cell todetect scattered light. The number of photodetectors can vary from asingle detector, generally located at 90° relative to the beamdirection, to a plurality of detectors located at various angles.

The flow distributor itself can take many forms. In a preferredembodiment of the invention, the flow distributor is a stainless steelwire mesh with a pore size of 25 μm. Experiments by the inventors haveverified that this configuration provides appreciable benefit to theuniform flow profile while minimizing the system back-pressure and thepossibility of plugging. Added benefits of the use of a stainless steelmesh as a flow distributor include its compatibility with both aqueousand organic solvents, durability, and low cost. The mesh at the inlettranslates a single orifice 1001 injecting sample into the cell intoplurality of orifices, each of which introduces a portion of the sampleinto the cell. The resulting flow entering the cell resembles aplurality of Poiseuille distributions at each tiny orifice, which, whenaveraged together approximate plug flow as it enters flow cell. The plugflow then heals back into a large-scale Poiseuille profile as the sampletraverses the measurement volume. A disc shaped frit may also act as aflow distributor.

In another embodiment of the invention, a spool shaped annular flowdistributor can be used. This is particularly useful for lightscattering measurements utilizing the parallel flow cell configuration;as a screen or frit will block the light beam passing along themeasurement bore in many configurations. An example of an embodiment ofa parallel cell MALS assembly in which the annular flow distributor isused is shown in FIG. 11(a), and a side and top view of the annulardistributor itself is shown in FIG. 11(b). In this configuration, thesample enters a retaining inlet manifold 1101, and flow is directed intothe annular distributor 1102, by means of a channel 1103 extendingaround the circumference of the distributor. The distributor's innersurface 1104 is porous, and tiny jets of fluid then enter the bore ofthe flow cell radially therefrom by first filling the empty region 1112of the distributor. The porous element 1104 can be made of a annularfrit or screen, or could be constructed as a solid annular block with aseries of radial holes drilled or etched in it. The annular distributoris contained by sealing means 1106 such as gaskets or o-rings bothbetween the cell structure 1105 and window 1107 through which the beam1108 passes and which is held into the manifold by a retaining ring1109. As the pressure required to fill the annular channel ΔP₂ is smallcompared to the pressure required to pass through the porous elements ofthe distributor ΔP₁, the condition ΔP₂<<ΔP₁ is satisfied and followingthe argument above, the sample passing thereinto is well mixed prior toentering the cell itself. The annular distributor is ideal for use witha parallel cell configuration. One could combine the annual flowdistributor with flat distributor similar to described above. Forexample, a frit element could be placed at a position near the junctionof the inlet tubing with the inlet manifold 1110 to further homogenizethe sample prior to entry into the region 1103. Alternatively theannular flow distributor could be used with a cell in the perpendicularconfiguration, and without the need for the sealing window to betransparent, indeed, in this case there is no need for a window at all,and the annular distributor can comprise a solid end 1111, as shown inFIG. 11c , permitting thereby, fluid flow only in one direction, namelyinto the cell. Additionally, the annular distributor could be positionedat the outlet of the sample cell with or without a flow distributor atthe inlet.

Another embodiment of the invention makes use of a novel flow disruptorplaced within the flow path, either at the entrance or exit (or both) ofthe measurement cell. A flow disruptor may take many forms, thepreferred embodiment of which is described in FIG. 12 wherein a solidblocking element 1203, made of a material which is chemically stable inthe solvent to be used in the chromatography system, disrupts theincoming jet of fluid into or out of the cell. This blocking element1203 is placed within a generally circular gasket 1204, usually made ofPolytetrafluoroethylene (PTFE), the material commonly referred to by thetrade name Teflon (The Chemours Company, Wilmington Del.). The blocking1203 element may preferably be made of a shape, such as a square or anequilateral triangle, with lateral dimensions chosen such that it can beseated, without moving in a lateral direction, within the opening of thegasket 1204. The gasket 1204 and blocking element 1203 form thereby awell-defined, restricted flow path through which sample may pass fromthe inlet tubing to the measurement cell. The gasket and blockingelement are sandwiched between two screen elements 1202 that arecomposed of a material which is inert in the solvent to be used in thechromatography system, such as stainless steel or nickel. The screens1202 provide relatively uniform fluid path and are capable of retainingthe sandwiched elements 1203 and 1204 in place along the flow path. Theresulting sandwich is further sandwiched between two more circular PTFEgaskets 1201, which may be of identical dimensions to gasket 1204, orwhich may vary such that the meet spatial requirements of a manifold inwhich the resulting layers are contained. FIG. 12(b) shows a top view ofthe assembled flow distribution system.

While any and all combinations of the means by which flow through amulti detector measurement cell described herein can be managed are partof the present invention, the following example sets forth a preferredembodiment combining several of the elements to optimize MALSmeasurements is shown in FIG. 13. In this embodiment a coiled section oftubing 1304 is connected by an inlet fitting which mounts to the topsection 1303 of a manifold comprised of two halves. Sandwiched betweenthe two manifold halves 1303 and 1308, is the optically transparentmeasurement cell 1302. The two halves of the manifold may be heldtogether by bolts, clamps or other means. Flow through the inlet tubing1304 passes through the inlet fitting and comes into contact with aninlet flow distributor 1305 which, in this embodiment is the flowdisruptor shown in FIG. 12. Flow passes from the inlet flow disruptor1305 into and through the measurement cell 1302 where it is illuminatedby a fine light beam 1301 such as is generated by a laser. About themeasurement cell a plurality of photodetectors are placed to measurelight scattered from the illuminated beam by the sample. Upon exitingthe measurement cell, the sample passes through the exit flow disruptor1306 and into the exit tubing 1307, which is held in place within themanifold element 1308 by an outlet fitting. Inlet and outlet fittingsare generally made from a material such as polyether ether ketone (PEEK)or stainless steel. Flow passing through the exit tubing 1307 may thenpass on to another measurement device in the chain which may or may notcontain similar fittings, coiled tubing and flow distributors ordisruptors. It should be noted that the inlet and outlet tubing is shownas a short length of tightly coiled tubing 1304 and 1307 respectively inFIG. 13, however various other configurations may exist for the coiledtubing, including tubing that is tightly wound, that is loosely wound,that is extended a significant distance, that acts only Poiseuille flowdisruptor (such as the small lengths shown in FIG. 13), or lengthscoiled for as much of the distance between the column and themeasurement cell as is reasonably possible. The same configurations arealso possible and may be used between the MALS instrument and the nextmeasurement instrument downstream from the MALS instrument. It shouldalso be recognized that it is not necessary, though it is oftenpreferable, that the MALS instrument be the first in line following theseparation system.

While the coiled tubing is generally composed of stainless steel, thisdisclosure is not limited to this material alone. Other materials whichcan retain a coiled shape or which can be made to maintain the coiledshape while still withstanding the pressure required in an HPLC, UHPLCor other separation system, such as FFF, may also be used. For example,PEEK tubing may be coiled and held into this coiled configuration by awire support external to the tubing. Further, while the preferred boresize for UHPLC measurements might be 100 μm, the disclosure should notbe considered limited to this size, but can include any standard orcustom chromatography tubing capable maintaining a coiled shape or beingmade to maintain a coiled shape. Possible inner sizes of this coiledtubing include 127 μm, 178 μm, 254 μm, 508 μm and 1016 μm as well astubing of variable inner bore diameter.

Measurement of detector lag and visualization of concentration contourlines

The measurement lag of concentration contours between non-aligneddetectors discussed above was experimentally demonstrated by injecting astandard UHPLC sample into a solvent stream and monitoring its passagethrough the system with a CCD camera at multiple scattering angles.Narrow strips along the breadth of the bore were monitored as the samplepassed there through, and the intensity at a plurality positions wasrecorded. The time delay was calculated based on the arrival time of theintensity maxima recorded at each position within the measurementvolume. The results of several runs can be seen in FIGS. 14-17. Thepresented data shows the resulting time delays, and provides avisualization of the sample front for several conditions.

FIG. 14 graphically demonstrates the effect of Dean flow on the sampleprofile in the measurement cell. Trace 1 shows the flow sample profilewith the inlet tubing bent slightly towards the laser beam entrance.Trace 2 shows the resultant sample profile wherein the inlet tubing isbent towards the forward monitor, opposite the laser. Trace 3 shows theprofile of tubing connected to the cell in as straight a manner aspossible. This data clearly indicates that small bends in the inlettubing can create dramatically different flow profiles in the cellitself, which will cause time delay issues for slightly misaligneddetectors discussed previously.

FIG. 15 illustrates a near ideal configuration wherein the inlet tubinginto the flow cell is perfectly straight, and therefore has no bendradius. The flow rate used was 0.3 mL/min, the inner diameter of theinlet tubing was 100 μm and the inlet tubing length was 250 mm. Astainless steel screen was used as a flow distributor. As can be seenfrom the data, the contour front is relatively symmetrical, althoughthere is still clear time dependence in the sample profile.

FIG. 16, by contrast, shows severe asymmetry to the sample profile. Asin the previous example, the flow rate used was 0.3 mL/min, the innerdiameter of the inlet tubing was 100 μm and the inlet tubing length was250 mm. A stainless steel screen was used as a flow distributor.However, in this example, the inlet tubing had a bend radius of 25 mmprior to entry into the cell. For this case the Dn²Sc parameter for the25 mm bend is approximately 3500, well above the minimum valueindicating tube curvature is impactful to peak dispersion.

Lastly, FIG. 17 shows data collected wherein, again the tubing enteringthe inlet of the cell has a bend radius of about 25 mm, however a coilis added to the tubing just before entrance into the cell and after the25 mm bend. Again, the flow rate used was 0.3 mL/min, the inner diameterof the inlet tubing was 0.004″ and the inlet tubing length was 250 mmwith a coil bend radius of 1.6 mm. A stainless steel screen was used asa flow distributor. As can be clearly seen, the addition of the coilwith a flow distributing screen returns the profile to the symmetricstate similar to that seen in FIG. 15, wherein the tubing wasimpractically straight. For this case the Dn²Sc parameter for the 1.6 mmcoil is approximately 67000, almost 20 times the value of the 25 mmbend. The much greater Dn²Sc value of the coil compared to the inletbend results in the dispersion created by the coil has a greaterinfluence on the sample flow within the tube resulting in a much moreuniform sample concentration, both radially and axially, before enteringthe sample cell.

FIG. 18 contrasts results of chromatogram traces with three detectorsthat are not aligned to the same sample volume within the flow cell. Thedetector traces have been normalized to the same amplitude to highlightthat small misalignment results in a time delay between detector traces,seen in FIG. 18a . A time delay of 50 msec between the detectors couldresults in a radius error of up to 10%. By contrast the data shown inFIG. 18b , where each detector is seeing a similar sample volume showsall three detector signals well aligned with no appreciable delay time,resulting in an accurate measurement of the sample radius.

As will be evident to those skilled in the arts of light scattering andthe characterization of macromolecules there are many obvious variationsof the methods and devices of our invention that do not depart from thefundamental elements that we have listed for their practice; all suchvariations are but obvious implementations of the invention describedhereinbefore and are included by reference to our claims, which follow.

What is claimed is:
 1. An apparatus comprising: a retaining inletmanifold configured to be connected to inlet tubing and configured toallow a liquid sample to flow from the inlet tubing, through theretaining inlet manifold, and into an inlet of a flow cell configured tobe viewed by detector elements; and an annular distributor comprising achannel extending around a circumference of the distributor, an innersurface, and an empty region, wherein the inner surface is porous,wherein the distributor is configured to allow the liquid sample to flowfrom the retaining inlet manifold, through the inner surface radiallyinto the empty region, and from the empty region into the inlet of theflow cell, resulting in a flow entering the flow cell, wherein the flowis well mixed as the flow enters the flow cell.
 2. The apparatus ofclaim 1 wherein the inner surface comprises an annular frit.
 3. Theapparatus of claim 1 wherein the inner surface comprises a screen. 4.The apparatus of claim 1 wherein the distributor comprises a series ofradial holes.
 5. The apparatus of claim 1 further comprising: a firstseal proximate to a surface of the flow cell and proximate to a firstside of the distributor, a second seal proximate to a second side of thedistributor and proximate to a first side of a window within themanifold, wherein the first seal and the second seal are configured tocontain the distributor in the manifold; and a retaining ring proximateto a second side of the window and configured to hold the window in themanifold.